Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
1999-06-02
2001-06-26
Whisenant, Ethan (Department: 1655)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C436S063000, C436S064000, C536S023100, C435S006120, C435S091100, C435S091200
Reexamination Certificate
active
06251682
ABSTRACT:
BACKGROUND OF THE INVENTION
Microtubules are a major filament of the cytoskeleton and are involved in various biologic phenomena including mitosis, cell shape determination, cell locomotion and movement of intracellular organelles. Tubulin is one of the major microtubular components. Polymerization and depolymerization of tubulin regulate microtubular dynamics. Microtubules are considered one of the most important molecular targets for cancer chemotherapy.
Antimitotic agents which disrupt microtubules can be classified into two categories based on mechanism of action. These are the vinca alkaloids such as estramustine, rhizoxin and E7010, which inhibit microtubule polymerization, and taxanes such as paclitaxel and docetaxel which promote polymerization of microtubules and enhance microtubule stability.
The anti-mitotic anticancer agent paclitaxel is active against solid tumors. Paclitaxel is a microtubule-disrupting agent that primarily targets tubulin. In the absence of guanosine triphosphate (GTP), paclitaxel induces polymerization and stabilizes tubulin to cold- or calcium-induced microtubule depolymerization, thereby blocking cell cycle in the M phase. Tubulin is a heterodimer that consists of the alpha- and beta-tubulin subunits that form the microtubule.
Clinical trials with taxanes such as paclitaxel and docetaxel have revealed these agents to be effective against several cancers which were advanced or resistant to other anticancer drugs, especially breast cancer, ovarian cancers and non small cell lung carcinoma (NSCLC). With respect to NSCLC, a number of randomized clinical trials have demonstrated that survival in patients with advanced stage III or IV NSCLC can be prolonged with paclitaxel. Preliminary studies with paclitaxel showed response rates of 21% and 24% and an impressive 1-year survival rate of 45% in one trial (Chang et al. J. Natl Cancer Inst. 85:388-394, 1993; and Murphy et al. J. Natl Cancer Inst. 85:384-388, 1993). Paclitaxel is now often used in combination with other cytotoxic drugs including cisplatin and carboplatin in patients with NSCLC.
However, the acquisition of drug-resistant tumor cells is still a major problem in the medical treatment of malignant disease. The hydrophobic nature of drugs such as paclitaxel is known to induce overexpression of the MDR1 gene (Horwitz et al. Monogr J. Natl Cancer Inst. 15:55-61, 1993). However, paclitaxel resistant human lung cancer cells selected in the presence of low levels of paclitaxel do not express MDR1 (Kavallaris et al. J. Clin, Invest. 100:1282-1293, 1997). Further, cells expressing high levels of the multidrug resistance (MDP)-associated protein MRP display no or low resistance to paclitaxel (Zamas et al. Proc. Natl Acad. Sci. USA 91:8822-8826 1994).
It has now been found that mechanisms of drug resistance to anti-cancer agents such as paclitaxel in cancer are related to mutations in the beta-tubulin gene which affect microtubule dynamics.
SUMMARY OF THE INVENTION
An object of the present invention is to provide prognostic markers for efficacy of anti-cancer agents in patients suffering from cancer, wherein the prognostic markers comprise mutant tubulin genes.
Another object of the present invention is to provide a method of prognosticating the efficacy of anti-cancer agents in a patient suffering from cancer which comprises obtaining a biological sample from the patient and analyzing the sample for mutant tubulin genes, wherein the presence of mutant tubulin genes is indicative of a resistance to anti-mitotic anti-cancer agents such as taxanes.
DETAILED DESCRIPTION OF THE INVENTION
Point mutations in the tubulin gene have now been linked with paclitaxel resistance in human tumors in the clinical setting. A group of patients without beta-tubulin gene mutations had a 39.4% response rate with a 10 month median survival time, and 1-year, 3-year and 5-year survival rates of 33.3%, 12.1% and 3%, respectively. In contrast, patients with beta-tubulin mutations showed no response to paclitaxel treatment and poor survival.
Specifically, forty-nine patients with non small cell lung carcinomas were examined. Forty-eight patients underwent biopsy before starting chemotherapy to obtain tissue for histologic diagnosis. A portion of the tumor specimen was also processed for genetic analysis. One patient had a biopsy performed after receiving paclitaxel. Forty-Three patients were treated with paclitaxel 210 mg/m
2
in a 3-hour infusion every 9 weeks. Six patients were treated with paclitaxel 200 mg/m
2
in a 24-hour infusion every 3 weeks. After three courses of paclitaxel, a decision was made regarding continuing treatment. All tumor responses were submitted to a peer-review process by two independent radiologists. Responders received up to a maximum of 10 courses. Responses were graded as complete if all evidence of disease disappeared on follow-up computed tomography scans. A partial response was defined as more than a 50% reduction in the sum of products at the largest perpendicular diameter of all indicator lesions. Survival was calculated from the date of first treatment to the most recent follow-up contact or to the date of death and included all patients in the study. Median follow-up from the time of treatment for the entire series of patients was 7 months (range 1 to 67 months).
The genomic DNA of exons 1 through 4 of the beta-tubulin gene was sequenced in all 49 patients. Findings from this analysis provided evidence for mutations. To distinguish somatin mutations from rare germline variants, variations in normal control DNA for each patient were also determined. Normal control DNA was obtained either: from nonepithelial normal tissue in archival paraffin-embedded biopsy samples; by isolation of DNA from distant normal nonepithelial, archival paraffin blocks other than the biopsy samples; or in six patients, by venipuncture and isolation of lymphocyte DNA. This analysis showed that patients' tumors contained true somatic mutations when matched with normal control DNA. Somatic mutations are shown in Table 1. Of the 19 somatic mutations identified in 16 patients, 13 were missense mutations, one was a single base-pair insertion, three were 1 base pair deletions and 2 were nonsense mutations. This analysis showed that 16 patients (33%; 95% CI, 20.7% to 45.3%) had beta-tubulin mutations.
There were no differences in baseline characteristics, including performance status and stage, in patients with and without beta-tubulin mutations. However, none of the patients with beta-tubulin mutations attained an objective response; one had stable disease and 15 had progressive disease. In contrast, of the remaining 33 patients without beta-tubulin mutations (including one patient who had a beta-tubulin polymorphism), 13 had partial or complete response (39.4%; 95% CI, 22.8% to 56%; p=0.01). Median survival for the 16 patients with beta-tubulin mutations was 3 months (95% CI, 2 to 3.9 months), whereas for the 33 patients with no beta-tubulin mutations, median survival was 10 months (95% CI, 7.9 to 12.1 months, p=0.0001). A set of monoclonal antibodies to detect and discriminate tubulin isotypes I, II and III showed no differences in histopathologic data, clinical data, or survival.
All patients with beta-tubulin mutations were chemotherapy naive, whereas in the group of patients with tubulin mutations, three North American patients had been unsuccessfully treated with cisplatin. Although one patient (Case A1 in Table 1) with beta-tubulin mutations survived for 23 months, the individual did not attain even a partial response. No second-line chemotherapy was foreseen for nonresponders, although 6 of the 14 patients with stage IIIB received radiotherapy after completion of the study.
TABLE 1
Beta-Tubulin Mutations Found in Non Small Cell Lung Carcinoma Patients
Patient/
Nucleotide
Amino acid
Follow-up
Stage
Exon
Codon
change
substitution
Mutant Type
(months)
Status
MS/IV
4
245
CAG→AAG
Gln→Lys
transversion
1
DOD
147
ATG→AT
−1G frameshift
deletion
V9/IV
4
245
CTC→CTCC
+1C fra
Monzó Mariano
Rosell Rafael
Bristol--Myers Squibb Company
Licata & Tyrrell P.C.
Lu Frank
Whisenant Ethan
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